The effect of Btk inhibitors on agonist-induced platelet aggregation and clot retraction in vitro. (A-D) Human PRP was normalized to 100 × 10⁹/L and incubated with a range of doses (0.25-1.0 µM) of ibrutinib, zanubrutinib, or vehicle control for 10 minutes at 37°C. Aggregation responses of PRP were determined after agonist stimulation by 1.0 μg/mL CRP (A), 1.0 μg/mL collagen (B), 5 μM ADP (C), and 25 μM TRAP (D). These results are represented as the mean ± SEM of 3 independent experiments. *P ≤ .05, ** P ≤ .01, *** P ≤ .005, **** P ≤ .001. Normalized PRP from a healthy human donor (100 × 10⁹/L platelets) was left untreated or treated with 0.5 μM ibrutinib or 0.5 µM zanubrutinib for 5 minutes at 37°C. Clot formation was induced with 2.5 U/mL of thrombin and monitored over time at 37°C. (E) Representative images of clot retraction using PRP, untreated or treated with 0.5 μM ibrutinib or 0.5 µM zanubrutinib after addition of 2.5 U/mL of thrombin over time. (F) The remaining serum volume was measured and recorded after clot retraction at 15, 30, 45, 60, and 120 minutes. Data are representative of 4 independent experiments and are expressed as the mean ± SEM, by Student t test. **P ≤ .001, ***P ≤ .0001.